Endothelial glycocalyx conditions influence nanoparticle uptake for passive targeting

Abstract

Cardiovascular diseases are facilitated by endothelial cell (EC) dysfunction and coincide with EC glycocalyx coat shedding. These diseases may be prevented by delivering medications to affected vascular regions using circulating nanoparticle (NP) drug carriers. The objective of the present study was to observe how the delivery of 10 nm polyethylene glycol-coated gold NPs (PEG-AuNP) to ECs is impacted by glycocalyx structure on the EC surface. Rat fat pad endothelial cells were chosen for their robust glycocalyx, verified by fluorescent immunolabeling of adsorbed albumin and integrated heparan sulfate (HS) chains. Confocal fluorescent imaging revealed a ~3 µm thick glycocalyx layer, covering 75% of the ECs and containing abundant HS. This healthy glycocalyx hindered the uptake of PEG-AuNP as expected because glycocalyx pores are typically 7 nm wide. Additional glycocalyx models tested included: a collapsed glycocalyx obtained by culturing cells in reduced protein media, a degraded glycocalyx obtained by applying heparinase III enzyme to specifically cleave HS, and a recovered glycocalyx obtained by supplementing exogenous HS into the media after enzyme degradation. The collapsed glycocalyx waŝ2 µm thick with unchanged EC coverage and sustained HS content. The degraded glycocalyx showed similar changes in EC thickness and coverage but its HS thickness was reduced to 0.7 µm and spanned only 10% of the original EC surface. Both dysfunctional models retained six- to sevenfold more PEG-AuNP compared to the healthy glycocalyx. The collapsed glycocalyx permitted NPs to cross the glycocalyx into intracellular spaces, whereas the degraded glycocalyx trapped the PEG-AuNP within the glycocalyx. The repaired glycocalyx model partially restored HS thickness to 1.2 µm and 44% coverage of the ECs, but it was able to reverse the NP uptake back to baseline levels. In summary, this study showed that the glycocalyx structure is critical for NP uptake by ECs and may serve as a passive pathway for delivering NPs to dysfunctional ECs.

Regeneration of glycocalyx by heparan sulfate and sphingosine 1-phosphate restores inter-endothelial communication

“…Previously, our group and others successfully replaced shed HS with commercial HS [18]or sulodexide [19, 20], a compound containing both heparin and dermatan sulfate. In other studies, a non-animal heparan sulfate-like polysaccharide, called rhamnan sulfate, was used as a vascular EC HS mimetic [17, 21–23].…”

“…In previous studies, described above, efficacy of HS regeneration was tested by examining transendothelial permeability and vascular inflammation as markers of HS-dependent cell function [18–20, 25, 30]. Few other important EC functions were tested [20].…”

Glycocalyx in Atherosclerosis-Relevant Endothelium Function and as a Therapeutic Target

“…In general, EDHF-mediated responses include an increase in the intracellular calcium concentration. This results in an endothelium-dependent hyperpolarization of smooth muscle cells, which evokes electrical coupling through myoendothelial junctions and accumulation of potassium ions in the intercellular space [ 32 ]. Additionally, the endothelium releases vasoconstrictive factors such as thromboxane and endothelin-1 [ 20 ].…”

“…Another well-characterized EC response to healthy blood flow is the tightening of junctional interconnections between neighboring ECs in order to (i) reinforce the barrier between the blood and the underlying tissue [ 32 , 33 ] and (ii) link neighboring ECs so that they can communicate (Fig. 1 ).…”

“…In favorable blood flow conditions, endothelium protection and functionality are governed by expression of the GCX [ 15 , 19 , 32 , 43 – 51 ] that acts as both a buffer and a force transmitter. The GCX is a negatively charged, heavily hydrated, polysaccharide mesh layer that coats ECs [ 3 , 50 ].…”